Cancer can be effectively treated using targeted therapy. There are increasing efforts to fulfill the promise of targeted therapy using antibodies, peptides, and small molecules that selectively affect cancer cells. The key to successful targeted therapy is to identify target molecules that play a unique role in tumor cells. Tumor cells have a broad spectrum of mutations and chromosomal rearrangements affecting gene products that play critical roles in the genesis and maintenance of tumors (Hanahan and Weinberg, 2000; Vogelstein and Kinzler, 2004). Some of these mutated gene products are shared among cancer cells from different origins and may be good targets for developing anti-cancer drugs with a high therapeutic index.
Leads for new cancer therapeutics often emerge from target-based in vitro screening. For example, compounds that interfere with post-translational modifications of RAS, such as Farensyl Transferase Inhibitors (FTIs) are in clinical trials. In addition, targeted kinase inhibitors, such as Gleevec (Capdeville et al., 2002) and Sorafenib (Wilhelm et al., 2006) have been effective in treating tumors using a targeted therapy approach.
Many proteins cannot be targeted with enzymatic, active-site inhibitors. However, in some cases, the oncogenic functions of these targets can be inhibited by small molecules that disrupt their interaction with other necessary protein partners or other aspects of the protein's function. Small molecules that disrupt the interaction between the tumor suppressor p53 and the oncoprotein MDM2, or between pro-apoptotic and anti-apoptotic bcl-2 family proteins, induce selective death in a subset of tumor cells (Fry and Vassilev, 2005). In a more challenging approach to restore a mutant protein's function, others have reported the identification of pharmacological chaperones that convert the conformation of mutant p53 into wild type (Foster et al., 1999).
A complementary approach to such target-based discovery strategies is synthetic lethal screening. Activation of multiple downstream signaling molecules is crucial to RAS-mediated tumorigenesis and tumor maintenance. Synthetic lethal screening is a strategy for revealing critical RAS-linked targets. (Hartwell et al., 1997). For a given mutation “A”, if there exists a second mutation “B” that is particularly lethal to the organism in the presence of A, mutation B is synthetic lethal with mutation A because the lethality requires the synthesis, or bringing together, of the two mutations. In synthetic lethal screening with oncogenic RAS, the second perturbation can be created by using a small molecule to alter the function of a target protein, rather than by introducing a mutation in the gene encoding the protein. (Stockwell, 2000). The existence of striking synthetic lethal interactions in genome-wide studies has been demonstrated in the work of Tong et al. with yeast deletion strains (Tong et al., 2001).
Classic yeast synthetic lethal screening involves cDNA library transformation, genome wide mutagenesis and colony-color assays in order to identify clones that are necessary for the survival of yeast cells with a given mutation (Barbour and Xiao, 2006); alternatively, use of gene deletion panels has been successful (Tong et al., 2001). Although these classic methods have been useful in elucidating functional genetic connectivity in model organisms, they are not as applicable to mammalian systems. Small molecules, and additionally RNAi libraries using siRNAs or shRNAs (Moffat and Sabatini, 2006), are more tractable than mutagenesis for carrying out synthetic lethal screening in cell culture systems.
Unlike synthetic lethal screening with mutations, synthetic lethal screening with small organic molecules does not provide direct information about the nature of the two mutations that cause synthetic lethality. The relevant target of each compound must ultimately be identified.
However, the use of small molecules in place of gene deletions offers a number of advantages that can overcome the lack of direct information about the target protein. First, compound addition can be varied in time and concentration, providing finer control of the perturbation to the target cell. Second, small molecules can induce a gain-of-function or affect just one domain of a protein, unlike deletion mutations or RNA interference (RNAi). Third, it is more straightforward to develop a chemotherapeutic reagent from a small molecule lead than from a gene deletion or RNAi reagent. For these reasons, the concept of synthetic lethal screening with small molecules is complementary to such studies with gene deletions or RNAi. Indeed, several groups have reported the discovery of small-molecules that are synthetic lethal to mutations such as p53 (Stockwell et al., 1999), ERBB2 (Fantin et al., 2002), p21 (Torrance et al., 2001), and DPC4 (Wang et al., 2006). Further optimization of such hit compounds may yield anti-cancer leads with high therapeutic indices.
Synthetic lethal screening using RAS oncogenes has been studied (Dolma et al., 2003). Considering its critical role in cancer development, mutant RAS has been the focus of much research (Barbacid, 1987; Malumbres and Barbacid, 2003; Shaw and Cantley, 2006). Because restoring GTPase activity to mutant RAS is a challenging task for small molecules, efforts have focused on the more feasible approach of inhibiting post-translational processing of the RAS proteins to inactivate their oncogenic signaling. The C-terminal four amino acids of RAS proteins, i.e. the CAAX motif, are conjugated to a fifteen carbon isoprenoid by farensyltransferase (FTase) in the endoplasmic reticulum. This allows RAS proteins to be anchored in the plasma membrane, which is essential for activity (Schafer et al., 1989; Schafer et al., 1990). Several ‘CAAX peptide mimetic’ compounds have been developed as FTIs and have shown promising results in pre-clinical mouse studies (Kohl et al., 1995; Nagasu et al., 1995). However, these compounds showed mixed results in clinical trials, possibly because KRAS and NRAS, whose mutations are more prevalent than HRAS in human cancer, can be conjugated to a twenty carbon isoprenoid modification by geranylgeranyltransferase upon inhibition of FTase activity (Rowell et al., 1997; Whyte et al., 1997).
An alternative approach using ‘S-farensyl cystein mimetic’ compounds, such as FTS, has been reported to work on all three RAS proteins and to be less cytotoxic than FTIs (Marom et al., 1995). These compounds were identified as inhibitors of isoprenylcysteine carboxyl methyltransferase (ICMT), another enzyme involved in RAS protein maturation. However, these compounds are reported to have the ‘off-target’ effect of dislodging membrane-bound RAS proteins, which leads to inhibition of RAS signal activation (Marciano et al., 1995; Marom et al., 1995). The anti-tumor effect of FTIs and FTSs is significant in pre-clinical experiments and research is on-going to improve the efficacy of these reagents in clinical settings. However, there has been debate as to whether inhibition of RAS post-translational processing will result in cancer-cell selective inhibition, given that other proteins are also subject to the same lipid modifications.
The compound erastin was found to display synthetic lethality with oncogenic RAS. As expected, the target of erastin is not RAS itself: erastin binds to voltage-dependant anion channels, a novel target for anti-cancer drugs to induce RAS-RAF-MEK-dependent oxidative, non-apoptotic cell death. (Yagoda et al., 2007).
Accordingly, there exists a significant need to screen, identify, and/or develop compounds and compositions that selectively target tumor cells, especially RAS-selective lethal compounds and compositions.